DDR2 Mutations in Squamous Cell Lung Cancer

ABSTRACT

Methods for treating patients with squamous cell lung cancer, including detecting the presence of mutations in the discoidin domain receptor 2 (DDR2) gene.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/294,068, filed Oct. 14, 2016, which is a divisional of U.S. patentapplication Ser. No. 13/875,079, filed May 1, 2013, now U.S. Pat. No.9,499,856, which is a continuation under 35 U.S.C. § 111 ofInternational Patent Application No. PCT/US2013/030292, filed on Mar.11, 2013, and claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/619,273, filed on Apr. 2, 2012. The entire contents of theforegoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. LC090577awarded by the Department of Defense and Grant No. T32CA09172 awarded bythe National Institutes of Health. The Government has certain rights inthe invention.

TECHNICAL FIELD

This invention relates to methods for treating patients with squamouscell lung cancer, including detecting the presence of mutations in thediscoidin domain receptor 2 (DDR2) gene.

BACKGROUND

Lung cancer is the leading cause of cancer-related mortality in theUnited States with over 157,000 deaths projected in 2010 (1). The morecommon type of lung cancer, non-small cell lung cancer (NSCLC), accountsfor 85% of cases and carries a grim prognosis with approximately 70% ofpatients presenting with advanced and often incurable disease at thetime of diagnosis (2).

Despite these statistics a great deal of progress has been made in thetargeted treatment of patients with NSCLC, largely due to thedevelopment of small molecule inhibitors of the epidermal growth factorreceptor (EGFR) tyrosine kinase (3-5). Patients who respond to EGFRkinase inhibitors are much more likely to have the adenocarcinomasubtype of NSCLC (6). Patients with the other principal subtype ofNSCLC, lung squamous cell cancer (lung SCC), very rarely respond tothese agents and few advances have been made in the treatment of thistype of lung cancer which comprises 25% of NSCLC. In addition to EGFR,several other promising therapeutic targets have been identified in thelaboratory such as EML4-ALK, KRAS and MET; drugs directed against theseproteins are being tested in clinical trials (7-10). However, it appearsthat these targets are likely limited to adenocarcinomas as well. Arecent report has suggested that targeting FGFR1 amplifications in SCCof the lung may be a promising therapeutic strategy, though FGFRinhibitors are not currently in clinical use for the treatment ofpatients with lung cancer (11).

SUMMARY

At least in part, the present invention is based on the discovery thatmutations in the DDR2 kinase gene identify a novel therapeutic target insquamous cell lung cancer, and can be used to select therapy forpatients whose tumors comprises cells harboring DDR2 mutations, e.g.,administration of tyrosine kinase inhibitors such as dasatinib.

Thus in a first aspect, the invention provides methods for selecting atreatment comprising administration of a tyrosine kinase inhibitor (TKI)for a subject diagnosed with lung cancer, e.g., non-small cell lungcancer (NSCLC), e.g., squamous cell carcinoma (SCC). The methods includedetermining a nucleic acid sequence of all or part of a discoidin domainreceptor 2 (DDR2) gene in a sample comprising nucleated cells from theSCC in the subject; determining an expected amino acid translation ofthe determined DDR2 sequence; and comparing the expected amino acidtranslation with a reference amino acid sequence, wherein the referenceamino acid sequence is a wild type DDR2 sequence; detecting the presenceof at least one amino acid variation relative to the reference aminoacid sequence; and selecting a treatment comprising administration of aTKI for the subject, based on the presence of at least one amino acidvariation relative to the reference sequence.

In a further aspect, the invention provides methods for predictingresponse to a treatment comprising administration of a tyrosine kinaseinhibitor (TKI) in a subject diagnosed with squamous cell carcinoma(SCC). The methods include determining a nucleic acid sequence of all orpart of a discoidin domain receptor 2 (DDR2) gene in a sample comprisingnucleated cells from the SCC in the subject; determining an expectedamino acid translation of the determined DDR2 sequence; and comparingthe expected amino acid translation with a reference amino acidsequence, wherein the reference amino acid sequence is a wild type DDR2sequence; detecting the presence of at least one amino acid variationrelative to the reference amino acid sequence; and selecting a treatmentcomprising administration of a TKI for the subject, based on thepresence of the at least one amino acid variation relative to thereference sequence.

In some embodiments of the methods described herein, the subject ishuman and the reference amino acid sequence comprises SEQ ID NO:1. Insome embodiments of the methods described herein, the reference sequenceis obtained from non-cancerous cells of the same subject.

In some embodiments of the methods described herein, the at least oneamino acid variation comprises a non-conservative amino acidsubstitution.

In some embodiments of the methods described herein, the at least oneamino acid variation is within a kinase domain (amino acids 563-849) ordiscoidin domain (amino acids 30-185) of DDR2.

In some embodiments of the methods described herein, the at least oneamino acid variation is L63V, I120M, D125Y, L239R, G253C, G5055, C580Y,I638F, T765P, G774E/V, or S768R. In some embodiments of the methodsdescribed herein, the variation is due to a mutation is shown in Tables7 or 8.

In some embodiments of the methods described herein, the methods includedetermining a nucleic acid sequence of a coding region of a discoidindomain receptor 2 (DDR2) gene.

In some embodiments of the methods described herein, the at least oneamino acid variation results in a decrease in expression levels,half-life, or kinase activity of the DDR2 protein.

In some embodiments of the methods described herein, the TKI isdasatinib, nilotinib, imatinib, or ponatinib. In some embodiments of themethods described herein, the TKI is dasatinib.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1a-c : Sequencing of squamous lung cancer samples identifiesrecurrent mutations in DDR2. (a) Schema depicted for the primary,secondary and validation screens for DDR2 mutations in squamous lungcancer samples. (b) Amino acid sequence of DDR2 with the positions ofthe identified mutations shown in the context of the known domainstructure of DDR2. (c) Homology alignment of DDR2 amino acid sequence.Shown are the amino acid sequences of human DDR2, listed as DDR2 HUMANresidues 54-264 (SEQ ID NO:4) and 468-786 (SEQ ID NO:5); mouse DDR2,listed as DDR2 MOUSE residues 54-264 (SEQ ID NO:6) and 468-786 (SEQ IDNO:7); and the closest homologs in zebrafish, shown as ASWUM4 DANREresidues 27-236 (SEQ ID NO:8) and 443-755 (SEQ ID NO:9) and C. elegans,shown as Q95ZV7 CAEEL residues 50-248 (SEQ ID NO:10) and 451-722 (SEQ IDNO:11). Degree of homology is indicated by the bar graphs under eachamino acid and the position of the novel DDR2 mutations indicated.

FIGS. 2a-d : Lung cancer cell lines with DDR2 mutations are sensitive todrugs and RNAi targeting DDR2. (a) Proliferation of A549, NCI-H2286,HCC-366 and NCI-H1703 grown for six days in the presence of variousconcentrations of dasatinib. Proliferation shown relative to untreatedcells at the same time point. Standard errors are shown for triplicatesamples. (b) Proliferation shown of NCI-H2286 and HCC-366 cell linesectopically expressing the T654M gatekeeper mutation in DDR2, labeled asDDR2*. Six day proliferation in the presence of dasatinib is shown asabove. For NCI-H2286 and HCC-366 the gatekeeper mutation is expressed incis with the DDR2 mutation found in the cell line. (c) Proliferationmeasured as above for NCI-H2286, HCC-366 and NCI-H1703 cells stablyexpressing sh-RNA vectors targeting either GFP or the 3′ UTR of DDR2(DDR2 sh-RNA-2) or the coding sequence of DDR2 (DDR2 sh-RNA-5).Proliferation is measured after four days in culture as compared today 1. Standard errors are shown for triplicate samples. Immunoblotshowing relative levels of DDR2 in the cell lines used in the experimentis shown in the inset. “G” indicates cells expressing shGFP, and “2” and“5” the numbered DDR2 targeted hairpins. (d) Four-day proliferation ofthe DDR2 mutant NCI-H2286 and HCC-366 cell lines stably expressingectopic DDR2 following knock-down of DDR2 by a sh-RNA targeting the 3′UTR of DDR2 (sh-RNA-2). Proliferation of triplicate samples is presentedas above relative to cells transduced with a sh-RNA targeting GFP.Protein levels of DDR2 are shown in the immunoblot below, “G” indicatessh-GFP, “2” indicates expression of sh-RNA 2 and “D” indicatesexpression of sh-RNA2 and DDR2.

FIGS. 2e-i : Squamous lung cancer cell lines harboring DDR2 mutationsare sensitive to tyrosine kinase inhibitor treatment. (e) Proliferationof A549, NCIH2286, HCC-366 and NCI-H1703 grown for six days in thepresence of various concentrations of imatinib. Proliferation ispresented relative to untreated cells at the same time point. Standarderrors are presented for triplicate samples. (f) Viability measured bytrypan blue exclusion in A549, NCI-H2286, HCC-366 and NCI-H1703 cellsgrown in the indicated concentrations of dasatinib. Data are presentedas viability relative to untreated cells at the same time point andrepresent an average of fifty independently acquired trypan blue imagesin each of three replicates with standard errors shown. (g)Proliferation of A549, NCIH2286, HCC-366 and NCI-H1703 cells grown forsix days in the presence of nilotinib. Data are presented as above. (h)Proliferation of A549, NCI-H2286, HCC-366 and NCI-H1703 cells grown inthe presence of AP24534. Data are presented as above. (i) Immunoblotshowing DDR2 expression from cell lines used in the experiment shown inFIG. 2c . DDR2* denotes the “gatekeeper” transgene.

FIG. 3: Xenografts of squamous lung cancer cell lines demonstrateanti-tumor effects of dasatinib in vivo. Athymic nu/nu mice wereinjected subcutaneously with A549, NCI-H1703, HCC-366 and NCI-H2286cells (n=10) and treated with dasatinib or vehicle for two weeksfollowing tumor formation. Depicted are measurements of tumor size inmice from each cohort. Tumors did not form in the mice injected withHCC-366 and these mice could not be analyzed further.

FIGS. 4a-d : Ectopic expression of DDR2 mutants leads to cellulartransformation which can be blocked by dasatinib or combination tyrosinekinase inhibitor treatment. (a) Results from soft agar assay in which3T3 fibroblasts expressing the L63V DDR2 mutation, the L858R EGFRmutation or the KRAS G12V mutation were plated in soft agar in thepresence of various concentrations of dasatinib. Colony number of sixindependent samples with standard errors is shown. (b) Proliferation atfour days of Ba/F3 cells expressing vector only or one of six DDR2mutations shown in cells grown in the presence of dasatinib. For thevector control cells are grown in the presence of IL-3 to maintainviability and in the case of the DDR2 mutants all cells are IL-3independent and cultured in the absence of IL-3. Proliferation is shownrelative to untreated cells at the same time point for triplicatesamples with standard errors. (c) Proliferation of Ba/F3 cellsexpressing DDR2 L63V co-cultured with 50 nM of nilotinib, AP24534 ordasatinib with or without 500 nM AZD0530. Proliferation is relative tountreated cells grown in parallel. (d) Immunoblots of DDR2 L63Vtransformed Ba/F3 cells treated for two days with the depictedconcentrations of AZD0530 (AZD) in addition to 50 nM nilotinib (N),AP24534 (AP) or dasatinib (D). The first lane is an untreated sample.Shown are immunoblots probed with antibodies against phospho-Src Y416,phospho-STAT5 Y694, FLAG-DDR2 and actin.

FIGS. 4e-h : Ectopic expression of DDR2 leads to cellular transformationwhich is sensitive to AP24534 treatment. (e) Colony formation in softagar of NIH-3T3 fibroblasts stably expressing the vector alone orwild-type or various mutant forms of DDR2. Colony numbers with standarderrors are shown for six independent samples. Expression of FLAG-taggedDDR2 by immunoblotting of the cells used in the transformation assay isshown in the inset and actin serves as a loading control. 3T3fibroblasts expressing the activating L858R mutation of EGFR (EGFR*) areused as a positive control. (f) Time to IL-3 independence is shown forBa/F3 cells stably expressing vector alone or wild-type or mutant formsof DDR2. Expression level of the transgenes and KD DDR2 (K608E) is shownas well as actin. The KD DDR2 was probed on a separate membrane which isindicated by the separation bar.

(g) Proliferation at four days of Ba/F3 cells expressing vector only orone of six DDR2 mutations is shown in cells grown in the presence ofimatinib. For the vector control the cells are grown in the presence ofIL-3 to maintain viability and in the case of the DDR2 mutants all cellsare IL-3 independent and cultured in the absence of IL-3. Proliferationis shown relative to untreated cells at the same time point fortriplicate samples with standard errors. (h) Experiment as above withAP24534.

FIGS. 4i-l : DDR2-transformed Ba/F3 cells maintain Src and STAT5phosphorylation and treatment of Ba/F3 cells expressing mutant forms ofDDR2 with nilotinib or AZD0530 results in minimal toxicity. (i)Proliferation at four days of Ba/F3 cells expressing vector only or oneof six DDR2 mutations is shown in cells grown in the presence ofnilotinib. For the vector control the cells are grown in the presence ofIL-3 to maintain viability and in the case of the DDR2 mutants all cellsare IL-3 independent and cultured in the absence of IL-3. Proliferationis shown relative to untreated cells at the same time point fortriplicate samples with standard errors. (j) Immunoblots showing thelevels of phospho-Src (Y416), phospho-STAT5 (Y694) and actin in Ba/F3cells expressing mutant forms of DDR2 or the vector alone from the celllines shown in Supplementary FIG. 3b . All DDR2 mutant lines are grownin the absence of IL-3 and the vector is shown in the presence andabsence of IL-3. (k) Experiment as above with Ba/F3 cells grown in thepresence of AZD0530. (1) Immunoblots of DDR2 L63V transformed Ba/F3cells treated for two days with the depicted concentrations of AZD0530,nilotinib (N), AP24534 (AP) or dasatinib (D). The first lane is anuntreated sample. Shown are immunoblots probed with antibodies againstphospho-Src, phospho-STAT5, FLAG-DDR2 and actin.

FIGS. 4m-o : Combination treatment with nilotinib, AP24534 or dasatiniband AZD0530 leads to increased killing of DDR2 transformed Ba/F3 cells.(m) Proliferation at four days of Ba/F3 cells expressing vector only orone of six DDR2 mutations is shown in cells grown in the presence of afixed concentration 4 of nilotinib and the depicted amounts of AZD0530.For the vector control the cells are grown in the presence of IL-3 tomaintain viability and in the case of the DDR2 mutants all cells areIL-3 independent and cultured in the absence of IL-3. Proliferation isshown relative to untreated cells at the same time point for triplicatesamples with standard errors. (n) Experiment performed as above withAP24534 and AZD0530. (o) Experiment performed as above with dasatiniband AZD0530.

FIGS. 5a-b : Radiographic response of a patient with a S768R DDR2mutation treated with dasatinib plus erlotinib. (a) CT scan images shownfrom a lung SCC patient who was treated with chemotherapy and later withdasatinib plus erlotinib. Serial CT scans are shown at the time ofinitiation of chemotherapy, initiation of study treatment with dasatiniband erlotinib and following two months of treatment with dasatinib pluserlotinib. (b) Top panel: Tumor dimension measurements from the subjectabove starting four months prior to chemotherapy treatment and extendingto the time at which combination therapy with dasatinib and erlotinibwas discontinued. Bottom panel: Bar graph depicting measured tumorvolume by RECIST criteria of the subject prior to chemotherapy,following chemotherapy and following two months of dasatinib pluserlotinib therapy.

DETAILED DESCRIPTION

Described herein is the identification of novel somatic mutations in thediscoidin domain receptor 2 (DDR2) tyrosine kinase gene at a frequencyof 3.8% (n=11) in a sample set of 290 squamous cell lung cancer samples.DDR2 is a receptor tyrosine kinase which binds collagen as itsendogenous ligand and has been previously shown to promote cellmigration, proliferation and survival when activated by ligand bindingand phosphorylation (12-18). DDR1 and DDR2 mutations have been reportedin several cancer specimens, including four DDR1 mutations (W385C,A496S, F866Y, F824W) and two DDR2 mutations in lung cancer (R105S andN456S), but these reports have not been confirmed in independent samplesand functional characterization of the mutations has not been reported(19-21). As demonstrated herein, DDR2 mutation status is associated withsensitivity to the tyrosine kinase inhibitor dasatinib or to sh-RNAmediated depletion of DDR2. Additionally, DDR2 mutations are oncogenicand their ability to transform cells can be blocked by dasatinibtreatment or by combination tyrosine kinase inhibitor treatment.Furthermore, a DDR2 kinase domain mutation was observed in a clinicaltrial subject with SCC of the lung who had a radiographic response tocombination therapy with erlotinib and dasatinib and who did not have anEGFR mutation. Together, these data indicate that DDR2 may be animportant therapeutic target in SCCs, and can be used to select therapyfor patients with SCC.

Definitions

As used herein, an “allele” is one of a pair or series of geneticvariants at a specific genomic location. A “response allele” is anallele that is associated with increased likelihood of response;heterozygosity is sufficient as these are gain-of-function. Where a SNPis biallelic, one allele (the “response allele”) will be associated withincreased likelihood of response, while the other allele is associatedwith average or decreased likelihood of response, or some variationthereof.

As used herein, “genotype” refers to the diploid combination of allelesfor a given genetic polymorphism. A homozygous subject carries twocopies of the same allele and a heterozygous subject carries twodifferent alleles.

Microsatellites (sometimes referred to as a variable number of tandemrepeats or VNTRs) are short segments of DNA that have a repeatedsequence, usually about 2 to 5 nucleotides long (e.g., CACACA), thattend to occur in non-coding DNA. Changes in the microsatellitessometimes occur during the genetic recombination of sexual reproduction,increasing or decreasing the number of repeats found at an allele,changing the length of the allele. Microsatellite markers are stable,polymorphic, easily analyzed and occur regularly throughout the genome,making them especially suitable for genetic analysis.

The term “chromosome” as used herein refers to a gene carrier of a cellthat is derived from chromatin and comprises DNA and protein components(e.g., histones). The conventional internationally recognized individualhuman genome chromosome numbering identification system is employedherein. The size of an individual chromosome can vary from one type toanother with a given multi-chromosomal genome and from one genome toanother. In the case of the human genome, the entire DNA mass of a givenchromosome is usually greater than about 100,000,000 base pairs.

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (either RNA or its translation product, a polypeptide). A genecontains a coding region and includes regions preceding and followingthe coding region (termed respectively “leader” and “trailer”). Thecoding region is comprised of a plurality of coding segments (“exons”)and intervening sequences (“introns”) between individual codingsegments.

The term “reference sequence” refers to a sequence that is present in asubject considered to be a reference or control subject. The referencesequence as used in some embodiments is a sequence that is present inthe majority of a population, i.e., the “wild-type” sequence. In someembodiments, the reference sequence is nucleic acid (e.g., genomic DNAor mRNA/cDNA) or amino acid. In some embodiments, the reference sequenceis a sequence in the same subject at an earlier time point, e.g., beforetreatment. In some embodiments, the reference sequence is obtained fromnon-cancerous cells of the same subject.

The term “probe” refers to an oligonucleotide. In some embodiments, aprobe is single stranded at the time of hybridization to a target. Asused herein, probes include primers, i.e., oligonucleotides that can beused to prime a reaction, e.g., a PCR reaction.

The term “label” or “label containing moiety” refers in a moiety capableof detection, such as a radioactive isotope or group containing same,and non-isotopic labels, such as enzymes, biotin, avidin, streptavidin,digoxygenin, luminescent agents, dyes, haptens, and the like.Luminescent agents, depending upon the source of exciting energy, can beclassified as radioluminescent, chemiluminescent, bioluminescent, andphotoluminescent (including fluorescent and phosphorescent). In someembodiments, a probe described herein is bound, e.g., chemically boundto label-containing moieties or can be suitable to be so bound. Theprobe can be directly or indirectly labeled.

The term “direct label probe” (or “directly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isdetectable without further reactive processing of hybrid. The term“indirect label probe” (or “indirectly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isfurther reacted in subsequent processing with one or more reagents toassociate therewith one or more moieties that finally result in adetectable entity.

The terms “target,” “DNA target,” or “DNA target region” refers to anucleotide sequence that occurs at a specific chromosomal location. Eachsuch sequence or portion is preferably at least partially, singlestranded (e.g., denatured) at the time of hybridization. When the targetnucleotide sequences are located only in a single region or fraction ofa given chromosome, the term “target region” is sometimes used. In someembodiments, targets for hybridization are derived from specimens whichinclude, but are not limited to, chromosomes or regions of chromosomesin normal, diseased or malignant human cells, either interphase or atany state of meiosis or mitosis, and either extracted or derived fromliving or postmortem tissues, organs or fluids; germinal cells includingsperm and egg cells, or cells from zygotes, fetuses, or embryos, orchorionic or amniotic cells, or cells from any other germinating body;cells grown in vitro, from either long-term or short-term culture, andeither normal, immortalized or transformed; inter- or intraspecifichybrids of different types of cells or differentiation states of thesecells; individual chromosomes or portions of chromosomes, ortranslocated, deleted or other damaged chromosomes, isolated by any of anumber of means known to those with skill in the art, includinglibraries of such chromosomes cloned and propagated in prokaryotic orother cloning vectors, or amplified in vitro by means well known tothose with skill; or any forensic material, including but not limited toblood, or other samples.

The term “hybrid” refers to the product of a hybridization procedurebetween a probe and a target.

The term “hybridizing conditions” has general reference to thecombinations of conditions that are employable in a given hybridizationprocedure to produce hybrids, such conditions typically involvingcontrolled temperature, liquid phase, and contact between a probe (orprobe composition) and a target. Conveniently and preferably, at leastone denaturation step precedes a step wherein a probe or probecomposition is contacted with a target. Guidance for performinghybridization reactions can be found in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (2003),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. Hybridization conditions referred toherein are a 50% formamide, 2×SSC wash for 10 minutes at 45° C. followedby a 2×SSC wash for 10 minutes at 37° C.

Calculations of “identity” between two sequences are performed usingmethods known in the art, e.g., as follows. The sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in one orboth of a first and a second nucleic acid sequence for optimal alignmentand non-identical sequences can be disregarded for comparison purposes).The length of a sequence aligned for comparison purposes is at least 30%(e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the lengthof the reference sequence. The nucleotides at corresponding nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

In some embodiments, the comparison of sequences and determination ofpercent identity between two sequences is accomplished using amathematical algorithm. In some embodiments, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package, using a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5.

As used herein, the term “substantially identical” is used to refer to afirst nucleotide sequence that contains a sufficient number of identicalnucleotides to a second nucleotide sequence such that the first andsecond nucleotide sequences have similar activities. Nucleotidesequences that are substantially identical are at least 80% (e.g., 85%,90%, 95%, 97% or more) identical.

The term “nonspecific binding DNA” refers to DNA which is complementaryto DNA segments of a probe, which DNA occurs in at least one otherposition in a genome, outside of a selected chromosomal target regionwithin that genome. An example of nonspecific binding DNA comprises aclass of DNA repeated segments whose members commonly occur in more thanone chromosome or chromosome region. Such common repetitive segmentstend to hybridize to a greater extent than other DNA segments that arepresent in probe composition.

Methods of Selecting Treatment

Described herein are a number of methods of selecting a treatmentcomprising administration of a tyrosine kinase inhibitor for a subjectwho has squamous cell lung cancer.

As used herein, “detecting a variant DDR2” includes obtaininginformation regarding the identity (i.e., of a specific nucleotide),presence or absence of one or more specific sequences or alleles in asubject. Detecting a variant DDR2 can, but need not, include obtaining asample comprising DNA from a subject, e.g., from an SCC tumor cell,and/or assessing the identity, presence or absence of a specificsequence or one or more genetic markers in the sample. The individual ororganization who detects the variant DDR2 need not actually carry outthe physical analysis of a sample from a subject; the methods caninclude using information obtained by analysis of the sample by a thirdparty. Thus the methods can include steps that occur at more than onesite. In some embodiments, a sample is obtained from a subject at afirst site, such as at a health care provider. In some embodiments, thesample is analyzed at the same or a second site, e.g., at a laboratoryor other testing facility.

In some embodiments, to detect a variant DDR2, a biological sample thatincludes nucleated cells (such as blood, a cheek swab or mouthwash) isprepared and analyzed for sequence or the presence or absence ofpre-selected markers. Such diagnoses may be performed by diagnosticlaboratories, or, alternatively, in some embodiments, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis. In some embodiments, diagnostic orprognostic tests are performed as described herein or using well knowntechniques, such as described in U.S. Pat. No. 5,800,998.

In some embodiments, results of these tests, and optionally interpretiveinformation, are returned to the subject, the health care provider or toa third party payor. The results can be used in a number of ways. Insome embodiments, the information is communicated to the tested subject,e.g., with a prognosis and optionally interpretive materials that helpthe subject understand the test results and prognosis. In someembodiments, the information is used, e.g., by a health care provider,to determine whether to administer a treatment comprising a TKI for SCC,or whether a subject should be assigned to a specific category, e.g., acategory associated with a specific disease, or with drug response ornon-response. In some embodiments, the information is used, e.g., by athird party payor such as a healthcare payer (e.g., insurance company orHMO) or other agency, to determine whether or not to reimburse a healthcare provider for services to the subject, or whether to approve theprovision of services to the subject. For example, the healthcare payermay decide to reimburse a health care provider for treatments comprisingadministration of a TKI for SCC if the subject has a variant of DDR2 andthis is likely to response to a TKI. The presence or absence of a DDR2variant, e.g., as described herein, in a subject may be ascertained byusing any of the methods described herein.

The methods can include obtaining a sample comprising cells from thesubject, e.g., cells from a SCC tumor biopsy, and determining thesequence of all or part of the DDR2 gene. In some embodiments, genomicDNA is sequenced; in some embodiments, mRNA is sequenced (optionallyafter conversion to cDNA). In some embodiments, rather than sequencingall or part of a gene, the identity of a single nucleotide isdetermined.

Squamous Cell Lung Cancer

Non-small-cell lung cancer (NSCLC) is the leading cause of cancermortality in the United States and worldwide (Higgins and Ettinger,Expert Rev Anticancer Ther 2009; 9:1365-1378). Non-small-cell lungcancer can be divided into three major histological subtypes: squamouscell carcinoma, adenocarcinoma (AC) and large cell carcinoma (LCC). Thevast majority of SCCs arise in subsegmental or larger bronchi and growcentrally toward the main bronchus, infiltrating the underlyingbronchial cartilage, lymph nodes, and adjacent lung parenchyma. Theother types of NSCLC (AC and LCC) are often peripherally located in thelungs. Although exemplified in SCC, the methods described herein couldbe used in the other types of lung cancer, e.g., SCLC and NSCLC.

A suspected case of lung cancer, e.g., SCC can be initially identifiedbased on symptoms, followed by the presence of findings consistent withlung cancer, e.g., SCC on imaging studies, e.g., chest radiographs,Magnetic Resonance Imaging (MRI), positron emission tomography (PET), orcomputed tomography (CT), and a confirmed diagnosis obtained byhistology, e.g., by sputum cytologic studies, bronchoscopy, or CT-guidedtransthoracic needle biopsy of the mass; selection of the method candepend on the location of the tumor.

DDR2

DDR2 is a receptor tyrosine kinase that activates intracellularsignaling pathways including Ras/MAPK, ERK and NF-kB upon stimulation byits endogenous ligand, extracellular collagen (Vogel et al., J BiolChem, 2000. 275(8):5779-84; Vogel et al., Mol Cell, 1997. 1(1):13-23;Vogel et al., Mol Cell Biol, 2001. 21(8):2906-17). DDR2 is thought to beimportant in regulating cell growth and survival, as a DDR2 null mouseexhibits dwarfism and infertility and a family of humans with germlinehypomophic DDR2 mutations have short limbs and a short stature (Bargalet al., Am J Hum Genet, 2009. 84(1): 80-4). DDR2 mutations have beendescribed as rare events in a variety of cancers including a singlereport in lung cancer, though the significance of these events isunknown. (Rikova et al., Cell, 2007. 131(6):1190-203; Tomasson et al.,Blood, 2008. 111(9):4797-808) Furthermore, DDR2 has been identified as atarget of the FDA-approved tyrosine kinase inhibitors imatinib,nilotinib and dasatinib, with dasatinib the most potent based on largescale phosphpopeptide screening assays. (Day et al., Eur J Pharmacol,2008. 599(1-3): 44-53; Davies, H., et al., Cancer Res, 2005. 65(17):7591-5)

In some embodiments, the methods include determining the sequence of allor part of the DDR2 gene in a sample comprising cells, e.g., tumorcells, from the subject, and comparing the sequence to a reference DDR2gene or mRNA sequence. In some embodiments, the methods includesequencing all or part of the gene. The sequence of the DDR2 gene isknown in the art and is provided at GenBank Accession No. NG_016290.1.In some embodiments, the methods include sequencing the coding region ofthe DDR2 gene; two transcript variants exist, variant (1) (GenBank Acc.No. NM_001014796.1) and variant (2) (GenBank Acc. No. NM_006182.2),which differs in the 5′ UTR compared to variant 1. Both encode the sameprotein, i.e., GenBank Acc. No. NP_001014796.1 or NP_006173.2, whichboth set forth the following sequence:

(SEQ ID NO: 1)   1MILIPRMLLV LFLLLPILSS AKAQVNPAIC RYPLGMSGGQ IPDEDITASS QWSESTAAKY  61GRLDSEEGDG AWCPEIPVEP DDLKEFLQID LHTLHFITLV GTQGRHAGGH GIEFAPMYKI 121NYSRDGTRWI SWRNRHGKQV LDGNSNPYDI FLKDLEPPIV ARFVRFIPVT DHSMNVCMRV 181ELYGCVWLDG LVSYNAPAGQ QFVLPGGSII YLNDSVYDGA VGYSMTEGLG QLTDGVSGLD 241DFTQTHEYHV WPGYDYVGWR NESATNGYIE IMFEFDRIRN FTTMKVHCNN MFAKGVKIFK 301EVQCYFRSEA SEWEPNAISF PLVLDDVNPS ARFVTVPLHH RMASAIKCQY HFADTWMMFS 361EITFQSDAAM YNNSEALPTS PMAPTTYDPM LKVDDSNTRI LIGCLVAIIF ILLAIIVIIL 421WRQFWQKMLE KASRRMLDDE MTVSLSLPSD SSMFNNNRSS SPSEQGSNST YDRIFPLRPD 481YQEPSRLIRK LPEFAPGEEE SGCSGVVKPV QPSGPEGVPH YAEADIVNLQ GVTGGNTYSV 541PAVTMDLLSG KDVAVEEFPR KLLTFKEKLG EGQFGEVHLC EVEGMEKFKD KDFALDVSAN 601QPVLVAVKML RADANKNARN DFLKEIKIMS RLKDPNIIHL LAVCITDDPL CMITEYMENG 661DLNQFLSRHE PPNSSSSDVR TVSYTNLKFM ATQIASGMKY LSSLNFVHRD LATRNCLVGK 721NYTIKIADFG MSRNLYSGDY YRIQGRAVLP IRWMSWESIL LGKFTTASDV WAFGVTLWET 781FTFCQEQPYS QLSDEQVIEN TGEFFRDQGR QTYLPQPAIC PDSVYKLMLS CWRRDTKNRP 841SFQEIHLLLL QQGDE

In some embodiments, all or part of the DDR2 nucleic acid sequence is“translated” into a predicted amino acid sequence based on known rulesof codon-amino acid specification. This predicted amino acid sequencecan be evaluated for the presence of one or more differences from thereference amino acid sequence. In some embodiments, the presence of amissense mutation, e.g., a non-conservative variant, as compared to awild-type reference (e.g., SEQ ID NO:1) in the amino acid sequenceindicates an increased likelihood of response to a TKI. In someembodiments, a variant amino acid sequence is detected directly, e.g.,using methods known in the art, such as methods using antibodies thatbind specifically to a variant of DDR2 protein but not to wild-type, orby direct sequencing of the protein, e.g., using mass spectrometryanalysis or other methods known in the art.

The methods can include the detection of specific mutations describedherein, e.g., as shown in FIG. 1B or 1C, that alter the primary sequenceof the DDR2 protein, e.g., L63V, I120M, D125Y, L239R, G253C, G5055,C580Y, I638F, T765P, G774E/V, and/or S768R.

Alternatively, the methods can include the detection of other mutationsthat alter the primary sequence of the DDR2 protein (e.g., missense ornon-conservative mutations), or that do not alter the primary sequenceof the DDR2 protein but affect levels of expression and/or half-life ofthe protein. In some embodiments, the methods include detectingmutations that alter the primary sequence of the DDR2 protein and affectkinase activity of the protein. Mutations that affect expression levels,half-life, or kinase activity can be identified readily by one of skillin the art, e.g., using assays known in the art and/or described herein.For example, in some embodiments a recombinant protein is produced usingknown molecular biological techniques, e.g., obtaining a wild type orreference sequence (e.g., genomic or cDNA), using mutagenesis (e.g.,site-directed mutagenesis) to alter the sequence to reflect themutation, and expressing the mutated protein in a cell, e.g., amammalian cell, and assaying for protein levels and/or activity of theprotein.

In some embodiments, the mutations are in the discoidin domain, e.g.,amino acids 30-185 or 32-184 of NP_001014796.1; or the catalytic/kinasedomain, e.g., amino acids 557-851 or 563-849 of NP_001014796.1.

As in preferred embodiments the methods described herein will beperformed on human subjects, the human DDR2 gene sequence has beenprovided as an example. As one of skill in the art will appreciate, ifthe methods are performed on subjects of other species, a reference DDR2sequence obtained from that species should be used. In some embodiments,to identify a DDR2 reference sequence, a biological sample that includesnon-cancerous nucleated cells (such as blood, a cheek swab, ormouthwash) is prepared and analyzed for sequence, or for the presence orabsence of preselected markers. In some embodiments, direct analysis ofDDR2 proteins is used to detect the presence of mutations, using samplescomprising DDR2 proteins from the subject, e.g., tissue samples, e.g.,from a tissue biopsy; in some embodiments, a SCC biopsy is used. Suchdeterminations can be performed using methods known in the art bydiagnostic laboratories, or, alternatively, diagnostic kits can bemanufactured and sold to health care providers or to private individualsfor self-diagnosis; such kits can include primers, probes, or antibodiesthat bind specifically to a mutation in DDR2. Diagnostic or prognostictests can be performed as described herein or using well knowntechniques, such as described in U.S. Pat. No. 5,800,998. The presenceor absence of a DDR2 variant in a subject may be ascertained by usingany of the methods described herein. In some cases, results of thesetests, and optionally interpretive information, can be returned to thesubject or the health care provider. Information gleaned from themethods described herein can also be used to select or stratify subjectsfor a clinical trial. For example, the presence of a selected allelicvariant described herein can be used to select or exclude a subject forparticipation in a clinical trial, e.g., a trial of a treatment for SCC,e.g., using a TKI.

The subject can be an adult, child, fetus, or embryo. In someembodiments, the sample is obtained prenatally, either from a fetus orembryo or from the mother (e.g., from fetal or embryonic cells in thematernal circulation). Methods and reagents are known in the art forobtaining, processing, and analyzing samples.

In some cases, the biological sample is processed for DNA isolation. Forexample, DNA in a cell or tissue sample can be separated from othercomponents of the sample. Cells can be harvested from a biologicalsample using standard techniques known in the art. For example, cellscan be harvested by centrifuging a cell sample and resuspending thepelleted cells. The cells can be resuspended in a buffered solution suchas phosphate-buffered saline (PBS). After centrifuging the cellsuspension to obtain a cell pellet, the cells can be lysed to extractDNA, e.g., gDNA. See, e.g., Ausubel et al., Current Protocols inMolecular Biology, eds., John Wiley & Sons (2003). The sample can beconcentrated and/or purified to isolate DNA. All samples obtained from asubject, including those subjected to any sort of further processing,are considered to be obtained from the subject. Routine methods can beused to extract genomic DNA from a biological sample, including, forexample, phenol extraction. Alternatively, genomic DNA can be extractedwith kits such as the QIAAMP® Tissue Kit (Qiagen, Chatsworth, Calif.)and the WIZARD® Genomic DNA purification kit (Promega).

The absence or presence of a variant DDR2 associated with TKISENSITIVITY as described herein can be determined using methods known inthe art. For example, gel electrophoresis, capillary electrophoresis,size exclusion chromatography, sequencing, and/or arrays can be used todetect the presence or absence of a variant DDR2. Amplification ofnucleic acids, where desirable, can be accomplished using methods knownin the art, e.g., PCR. In one example, a sample (e.g., a samplecomprising genomic DNA), is obtained from a subject. The DNA in thesample is then examined to identify a variant DDR2 as described herein.The presence of the variant DDR2 can be determined by any methoddescribed herein, e.g., by sequencing or by hybridization of the gene inthe genomic DNA, RNA, or cDNA to a nucleic acid probe, e.g., a DNA probe(which includes cDNA and oligonucleotide probes) or an RNA probe. Thenucleic acid probe can be designed to specifically or preferentiallyhybridize with a particular variant.

Other methods of nucleic acid analysis can include direct manualsequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995(1988); Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977);Beavis et al., U.S. Pat. No. 5,288,644); automated fluorescentsequencing; single-stranded conformation polymorphism assays (SSCP)(Schafer et al., Nat. Biotechnol. 15:33-39 (1995)); clamped denaturinggel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE orTDGE); conformational sensitive gel electrophoresis (CSGE); denaturinggradient gel electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad.Sci. USA 86:232-236 (1989)); denaturing high performance liquidchromatography (DHPLC, Underhill et al., Genome Res. 7:996-1005 (1997));infrared matrix-assisted laser desorption/ionization (IR-MALDI) massspectrometry (WO 99/57318); mobility shift analysis (Orita et al., Proc.Natl. Acad. Sci. USA 86:2766-2770 (1989)); restriction enzyme analysis(Flavell et al., Cell 15:25 (1978); Geever et al., Proc. Natl. Acad.Sci. USA 78:5081 (1981)); quantitative real-time PCR (Raca et al., GenetTest 8(4):387-94 (2004)); heteroduplex analysis; chemical mismatchcleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401(1985)); RNase protection assays (Myers et al., Science 230:1242(1985)); use of polypeptides that recognize nucleotide mismatches, e.g.,E. coli mutS protein; allele-specific PCR, and combinations of suchmethods. See, e.g., Gerber et al., U.S. Pat. Publication No.2004/0014095 which is incorporated herein by reference in its entirety.

Sequence analysis can also be used to detect specific polymorphicvariants. For example, polymorphic variants can be detected bysequencing exons, introns, 5′ untranslated sequences, or 3′ untranslatedsequences. A sample comprising DNA or RNA is obtained from the subject.PCR or other appropriate methods can be used to amplify a portionencompassing the polymorphic site, if desired. The sequence is thenascertained, using any standard method, and the presence of apolymorphic variant is determined. Real-time pyrophosphate DNAsequencing is yet another approach to detection of polymorphisms andpolymorphic variants (Alderborn et al., Genome Research 10(8):1249-1258(2000)). Additional methods include, for example, PCR amplification incombination with denaturing high performance liquid chromatography(dHPLC) (Underhill et al., Genome Research 7(10):996-1005 (1997)).

In some embodiments, the methods described herein include determiningthe sequence of the entire region of the DDR2 locus described herein asbeing of interest. For example, a method provided herein can includedetermining a nucleic acid sequence of a DDR2 gene in a sample from ahuman subject, determining an expected amino acid translation of nucleicacid sequence; and comparing the expected amino acid translation with areference amino acid sequence. In such a method, the presence of atleast one amino acid variant (e.g., a non-conservative amino acidsubstitution) relative to the reference amino acid sequence can beindicative of TKI sensitivity or an increased likelihood of response toa TKI, or emergence of resistance to a TKI, in the human subject. Forexample, an amino acid variant can comprise a non-conservativesubstitution described herein. In some embodiments, the sequence isdetermined on both strands of DNA.

In order to detect sequence variants, it may be desirable to amplify aportion of genomic DNA (gDNA) or cDNA encompassing the variant site.Such regions can be amplified and isolated by PCR using oligonucleotideprimers designed based on genomic and/or cDNA sequences that flank thesite. PCR refers to procedures in which target nucleic acid (e.g.,genomic DNA) is amplified in a manner similar to that described in U.S.Pat. No. 4,683,195, and subsequent modifications of the proceduredescribed therein. Generally, sequence information from the ends of theregion of interest or beyond are used to design oligonucleotide primersthat are identical or similar in sequence to opposite strands of apotential template to be amplified. See e.g., PCR Primer: A LaboratoryManual, Dieffenbach and Dveksler, (Eds.); McPherson et al., PCR Basics:From Background to Bench (Springer Verlag, 2000); Mattila et al.,Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods andApplications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202. Other amplification methods thatmay be employed include the ligase chain reaction (LCR) (Wu and Wallace,Genomics 4:560 (1989), Landegren et al., Science 241:1077 (1988),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA86:1173 (1989)), self-sustained sequence replication (Guatelli et al.,Proc. Nat. Acad. Sci. USA 87:1874 (1990)), and nucleic acid basedsequence amplification (NASBA). Guidelines for selecting primers for PCRamplification are well known in the art. See, e.g., McPherson et al.,PCR Basics: From Background to Bench, Springer-Verlag, 2000. A varietyof computer programs for designing primers are available, e.g., ‘Oligo’(National Biosciences, Inc, Plymouth, Minn.), MacVector (Kodak/IBI), andthe GCG suite of sequence analysis programs (Genetics Computer Group,Madison, Wis. 53711).

In some cases, PCR conditions and primers can be developed that amplifya product only when the variant allele is present or only when the wildtype allele is present (MSPCR or allele-specific PCR). For example,subject DNA and a control can be amplified separately using either awild type primer or a primer specific for the variant allele. Each setof reactions is then examined for the presence of amplification productsusing standard methods to visualize the DNA. For example, the reactionscan be size-separated by agarose gel electrophoresis and the DNAvisualized by staining with ethidium bromide or other DNA intercalatingdye. In DNA samples from heterozygous subjects, reaction products wouldbe detected in each reaction.

Real-time quantitative PCR can also be used to determine copy number.Quantitative PCR permits both detection and quantification of specificDNA sequence in a sample as an absolute number of copies or as arelative amount when normalized to DNA input or other normalizing genes.A key feature of quantitative PCR is that the amplified DNA product isquantified in real-time as it accumulates in the reaction after eachamplification cycle. Methods of quantification can include the use offluorescent dyes that intercalate with double-stranded DNA, and modifiedDNA oligonucleotide probes that fluoresce when hybridized with acomplementary DNA.

In some embodiments, a peptide nucleic acid (PNA) probe can be usedinstead of a nucleic acid probe in the hybridization methods describedabove. PNA is a DNA mimetic with a peptide-like, inorganic backbone,e.g., N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T orU) attached to the glycine nitrogen via a methylene carbonyl linker(see, e.g., Nielsen et al., Bioconjugate Chemistry, The AmericanChemical Society, 5:1 (1994)). The PNA probe can be designed tospecifically hybridize to a nucleic acid comprising a polymorphicvariant conferring TKI sensitivity or resistance.

In some cases, allele-specific oligonucleotides can also be used todetect the presence of a variant. For example, variants can be detectedby performing allele-specific hybridization or allele-specificrestriction digests. Allele specific hybridization is an example of amethod that can be used to detect sequence variants, including completehaplotypes of a subject (e.g., a mammal such as a human). See Stonekinget al., Am. J. Hum. Genet. 48:370-382 (1991); and Prince et al., GenomeRes. 11:152-162 (2001). An “allele-specific oligonucleotide” (alsoreferred to herein as an “allele-specific oligonucleotide probe”) is anoligonucleotide that is specific for a particular polymorphism can beprepared using standard methods (see Ausubel et al., Current Protocolsin Molecular Biology, supra). Allele-specific oligonucleotide probestypically can be approximately 10-50 base pairs, preferablyapproximately 15-30 base pairs, that specifically hybridize to a nucleicacid region that contains a polymorphism. Hybridization conditions areselected such that a nucleic acid probe can specifically bind to thesequence of interest, e.g., the variant nucleic acid sequence. Suchhybridizations typically are performed under high stringency as somesequence variants include only a single nucleotide difference. In somecases, dot-blot hybridization of amplified oligonucleotides withallele-specific oligonucleotide (ASO) probes can be performed. See, forexample, Saiki et al., Nature (London) 324:163-166 (1986).

In some embodiments, allele-specific restriction digest analysis can beused to detect the existence of a variant, if the variants result in thecreation or elimination of a restriction site. Allele-specificrestriction digests can be performed in the following manner. A samplecontaining genomic DNA is obtained from the individual and genomic DNAis isolated for analysis. For nucleotide sequence variants thatintroduce a restriction site, restriction digest with the particularrestriction enzyme can differentiate the alleles. In some cases,polymerase chain reaction (PCR) can be used to amplify a regioncomprising the polymorphic site, and restriction fragment lengthpolymorphism analysis is conducted (see Ausubel et al., CurrentProtocols in Molecular Biology, supra). The digestion pattern of therelevant DNA fragment indicates the presence or absence of a variant andis therefore indicative of the presence or absence of TKI sensitivity orresistance. For sequence variants that do not alter a common restrictionsite, mutagenic primers can be designed that introduce a restrictionsite when the variant allele is present or when the wild type allele ispresent. For example, a portion of a nucleic acid can be amplified usingthe mutagenic primer and a wild type primer, followed by digest with theappropriate restriction endonuclease.

In some embodiments, fluorescence polarization template-directeddye-terminator incorporation (FP-TDI) is used to determine which ofmultiple polymorphic variants of a polymorphism is present in a subject(Chen et al., Genome Research 9(5):492-498 (1999)). Rather thaninvolving use of allele-specific probes or primers, this method employsprimers that terminate adjacent to a variant site, so that extension ofthe primer by a single nucleotide results in incorporation of anucleotide complementary to the polymorphic variant at the polymorphicsite.

In some cases, DNA containing an amplified portion may be dot-blotted,using standard methods (see Ausubel et al., Current Protocols inMolecular Biology, supra), and the blot contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the DNA is then detected. Specific hybridization of anallele-specific oligonucleotide probe to DNA from the subject isindicative of TKI resistance or sensitivity.

The methods can include determining the genotype of a subject withrespect to both copies of the polymorphic site present in the genome.For example, the complete genotype may be characterized as −/−, as −/+,or as +/+, where a minus sign indicates the presence of the reference orwild type sequence at the polymorphic site, and the plus sign indicatesthe presence of a variant other than the reference sequence. If multiplevariants exist at a site, this can be appropriately indicated byspecifying which ones are present in the subject. Any of the detectionmeans described herein can be used to determine the genotype of asubject with respect to one or both copies of the variant present in thesubject's genome.

Methods of nucleic acid analysis to detect variants can include, e.g.,microarray analysis. Hybridization methods, such as Southern analysis,Northern analysis, or in situ hybridizations, can also be used (seeAusubel et al., Current Protocols in Molecular Biology, eds., John Wiley& Sons (2003)). To detect microdeletions, fluorescence in situhybridization (FISH) using DNA probes that are directed to a putativelydeleted region in a chromosome can be used. For example, probes thatdetect all or a part of a microsatellite marker can be used to detectmicrodeletions in the region that contains that marker.

In some embodiments, it is desirable to employ methods that can detectthe presence of multiple variants (e.g., variants at a plurality ofsites) in parallel or substantially simultaneously. Oligonucleotidearrays represent one suitable means for doing so. Other methods,including methods in which reactions (e.g., amplification,hybridization) are performed in individual vessels, e.g., withinindividual wells of a multi-well plate or other vessel can also beperformed so as to detect the presence of multiple variants (e.g.,variants at a plurality of polymorphic sites) in parallel orsubstantially simultaneously.

Nucleic acid probes can be used to detect and/or quantify the presenceof a particular target nucleic acid sequence within a sample of nucleicacid sequences, e.g., as hybridization probes, or to amplify aparticular target sequence within a sample, e.g., as a primer. Probeshave a complimentary nucleic acid sequence that selectively hybridizesto the target nucleic acid sequence. In order for a probe to hybridizeto a target sequence, the hybridization probe must have sufficientidentity with the target sequence, i.e., at least 70% (e.g., 80%, 90%,95%, 98% or more) identity to the target sequence. The probe sequencemust also be sufficiently long so that the probe exhibits selectivityfor the target sequence over non-target sequences. For example, theprobe will be at least 20 (e.g., 25, 30, 35, 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or more) nucleotides in length. In someembodiments, the probes are not more than 30, 50, 100, 200, 300, 500,750, or 1000 nucleotides in length. Probes are typically about 20 toabout 1×10⁶ nucleotides in length. Probes include primers, whichgenerally refers to a single-stranded oligonucleotide probe that can actas a point of initiation of template-directed DNA synthesis usingmethods such as polymerase chain reaction (PCR), ligase chain reaction(LCR), etc., for amplification of a target sequence.

Control probes can also be used. For example, a probe that binds a lessvariable sequence, e.g., repetitive DNA associated with a centromere ofa chromosome, can be used as a control. Probes that hybridize withvarious centromeric DNA and locus-specific DNA are availablecommercially, for example, from Vysis, Inc. (Downers Grove, Ill.),Molecular Probes, Inc. (Eugene, Oreg.), or from Cytocell (Oxfordshire,UK). Probe sets are available commercially such from Applied Biosystems,e.g., the Assays-on-Demand SNP kits. Alternatively, probes can besynthesized, e.g., chemically or in vitro, or made from chromosomal orgenomic DNA through standard techniques. For example, sources of DNAthat can be used include genomic DNA, cloned DNA sequences, somatic cellhybrids that contain one, or a part of one, human chromosome along withthe normal chromosome complement of the host, and chromosomes purifiedby flow cytometry or microdissection. The region of interest can beisolated through cloning, or by site-specific amplification via PCR.See, for example, Nath and Johnson, Biotechnic. Histochem. 73(1):6-22(1998); Wheeless et al., Cytometry 17:319-326 (1994); and U.S. Pat. No.5,491,224.

In some embodiments, the probes are labeled, e.g., by direct labeling,with a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. A directly labeled fluorophoreallows the probe to be visualized without a secondary detectionmolecule. After covalently attaching a fluorophore to a nucleotide, thenucleotide can be directly incorporated into the probe with standardtechniques such as nick translation, random priming, and PCR labeling.Alternatively, deoxycytidine nucleotides within the probe can betransaminated with a linker. The fluorophore then is covalently attachedto the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No.5,491,224.

Fluorophores of different colors can be chosen such that each probe in aset can be distinctly visualized. For example, a combination of thefollowing fluorophores can be used: 7-amino-4-methylcoumarin-3-aceticacid (AMCA), TEXAS RED™ (Molecular Probes, Inc., Eugene, Oreg.),5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B,5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and CASCADE™blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). Fluorescentlylabeled probes can be viewed with a fluorescence microscope and anappropriate filter for each fluorophore, or by using dual or tripleband-pass filter sets to observe multiple fluorophores. See, forexample, U.S. Pat. No. 5,776,688. Alternatively, techniques such as flowcytometry can be used to examine the hybridization pattern of theprobes. Fluorescence-based arrays are also known in the art.

In other embodiments, the probes can be indirectly labeled with, e.g.,biotin or digoxygenin, or labeled with radioactive isotopes such as ³²Pand ³H. For example, a probe indirectly labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard colorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Oligonucleotide probes that exhibit differential or selective binding topolymorphic sites may readily be designed by one of ordinary skill inthe art. For example, an oligonucleotide that is perfectly complementaryto a sequence that encompasses a polymorphic site (i.e., a sequence thatincludes the polymorphic site, within it or at one end) will generallyhybridize preferentially to a nucleic acid comprising that sequence, asopposed to a nucleic acid comprising an alternate polymorphic variant.

In another aspect, the invention features arrays that include asubstrate having a plurality of addressable areas, and methods of usingthem. At least one area of the plurality includes a nucleic acid probethat binds specifically to a sequence comprising a variant describedherein, and can be used to detect the absence or presence of saidvariant, e.g., one or more SNPs, microsatellites, minisatellites, orindels, as described herein, e.g., to determine a haplotype comprising aplurality of variants. For example, the array can include one or morenucleic acid probes that can be used to detect a variant describedherein. In some embodiments, the array further includes at least onearea that includes a nucleic acid probe that can be used to specificallydetect another marker associated with TKI resistance or sensitivity, asdescribed herein. In some embodiments, the probes are nucleic acidcapture probes.

Generally, microarray hybridization is performed by hybridizing anucleic acid of interest (e.g., a nucleic acid encompassing apolymorphic site) with the array and detecting hybridization usingnucleic acid probes. In some cases, the nucleic acid of interest isamplified prior to hybridization. Hybridization and detecting aregenerally carried out according to standard methods. See, e.g.,Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S.Pat. No. 5,424,186. For example, the array can be scanned to determinethe position on the array to which the nucleic acid hybridizes. Thehybridization data obtained from the scan is typically in the form offluorescence intensities as a function of location on the array.

Arrays can be formed on substrates fabricated with materials such aspaper, glass, plastic (e.g., polypropylene, nylon, or polystyrene),polyacrylamide, nitrocellulose, silicon, optical fiber, or any othersuitable solid or semisolid support, and can be configured in a planar(e.g., glass plates, silicon chips) or three dimensional (e.g., pins,fibers, beads, particles, microtiter wells, capillaries) configuration.Methods for generating arrays are known in the art and include, e.g.,photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;5,510,270; and 5,527,681), mechanical methods (e.g., directed-flowmethods as described in U.S. Pat. No. 5,384,261), pin-based methods(e.g., as described in U.S. Pat. No. 5,288,514), and bead-basedtechniques (e.g., as described in PCT US/93/04145). The array typicallyincludes oligonucleotide probes capable of specifically hybridizing todifferent polymorphic variants. Oligonucleotide probes forming an arraymay be attached to a substrate by any number of techniques, including,without limitation, (i) in situ synthesis (e.g., high-densityoligonucleotide arrays) using photolithographic techniques; (ii)spotting/printing at medium to low density on glass, nylon ornitrocellulose; (iii) by masking, and (iv) by dot-blotting on a nylon ornitrocellulose hybridization membrane. Oligonucleotides also can benon-covalently immobilized on a substrate by hybridization to anchors,by means of magnetic beads, or in a fluid phase such as in microtiterwells or capillaries.

Arrays can include multiple detection blocks (i.e., multiple groups ofprobes designed for detection of particular variants). Such arrays canbe used to analyze multiple different variants. Detection blocks may begrouped within a single array or in multiple, separate arrays so thatvarying conditions (e.g., conditions optimized for particular variants)may be used during the hybridization. For example, it may be desirableto provide for the detection of those variants that fall within G-C richstretches of a genomic sequence, separately from those falling in A-Trich segments. General descriptions of using oligonucleotide arrays fordetection of variants can be found, for example, in U.S. Pat. Nos.5,858,659 and 5,837,832. In addition to oligonucleotide arrays, cDNAarrays may be used similarly in certain embodiments of the invention.

The methods described herein can include providing an array as describedherein; contacting the array with a sample (e.g., a portion of genomicDNA that includes at least a portion of human chromosome 14q32 (e.g., aregion between SNPs rs3783397 and r56576201) and/or optionally, adifferent portion of genomic DNA (e.g., a portion that includes adifferent portion of a human chromosome (e.g., including another regionassociated with TKI resistance or sensitivity)), and detecting bindingof a nucleic acid from the sample to the array. Optionally, the methodincludes amplifying nucleic acid from the sample, e.g., genomic DNA thatincludes at least a portion of an IFN2 gene, and, optionally, a regionthat includes another region associated with TKI resistance orsensitivity, prior to or during contact with the array.

In some aspects, the methods described herein can include using an arraythat can ascertain differential expression patterns or copy numbers ofone or more genes in samples from normal and affected individuals (see,e.g., Redon et al., Nature 444(7118):444-54 (2006)). For example, arraysof probes to a marker described herein can be used to measure variantsbetween DNA from a subject having TKI sensitive SCC and control DNA,e.g., DNA obtained from an individual that does not have TKI sensitiveSCC. Since the clones on the array contain sequence tags, theirpositions on the array are accurately known relative to the genomicsequence. Different hybridization patterns between DNA from anindividual with TKI sensitive SCC and DNA from a normal individual atareas in the array corresponding to markers in the human chromosome14q32 locus as described herein, and, optionally, one or more otherregions associated with TKI sensitive SCC, are indicative of sensitivityto the TKI. Methods for array production, hybridization, and analysisare described, e.g., in Snijders et al., Nat. Genet. 29:263-264 (2001);Klein et al., Proc. Natl Acad. Sci. USA 96:4494-99 (1999); Albertson etal., Breast Cancer Res. and Treatment 78:289-298 (2003); and Snijders etal. “BAC microarray based comparative genomic hybridization,” in Zhao etal. (eds), Bacterial Artificial Chromosomes: Methods and Protocols,Methods in Molecular Biology, Humana Press, 2002.

Tyrosine Kinase Inhibitors (TKIs)

Upon identification of a subject as having a tumor with one or more DDR2mutations as described herein, the methods can include administering atherapeutically effective amount of one or more tyrosine kinaseinhibitors. TKIs useful in the methods described herein can includeAxitinib (INLYTA; Pfizer), Critozinib (XALKORI; Pfizer), Dasatinib(SPRYCEL; BMS), Erlotinib (TARCEVA; Roche, Astellas), Gefitinib (IRESSA;Astra Zeneca), Imatinib (GLEEVEC; Novartis), Lapatinib (TYKERB; GSK),Nilotinib (TASIGNA; Novartis), Pazopanib (VOTRIENT; GSK), Ruxolitinib(JAKAFI; Incyte, Novartis), Sorafenib (NEXAVAR; Bayer/Onyx), Sunitinib(SUTENT; Pfizer), Vandetanib (CAPRELSA/ZACTIMA; Astra Zeneca), ponatinib(AP24534; ARIAD); Vemurafanib (ZELBORAF; Roche/Daiichi Sankyo),lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416),vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248),and leflunomide (SU101). In preferred embodiments, the TKI is dasatinib,nilotinib, imatinib, and/or ponatinib.

SPRYCEL (dasatinib, Bristol-Myers Squibb [BMS]-354825) is a potent,broad spectrum inhibitor of 5 critical oncogenic tyrosine kinases/kinasefamily members (BCR-ABL, SRC, c-KIT, PDGF receptor β [PDGFRβ], andephrin [EPH] receptor kinases), each of which are activated in multipleforms of human malignancies, and was discovered and developed by BMS.The chemical name for dasatinib isN-(2¬chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4¬pyrimidinyl]amino]-5-thiazolecarboxamide,monohydrate. The molecular formula is C₂₂H₂₆ClN₇O₂S.H₂O, whichcorresponds to a formula weight of 506.02 (monohydrate). The anhydrousfree base has a molecular weight of 488.01. SPRYCEL is approved in theUnited States (US)i, Europe (EU), and several other countries for thetreatment of adults in all phases of chronic myeloid leukemia (CML) withresistance or intolerance to prior therapy including imatinib, and inpatients with Philadelphia chromosome positive acute lymphoblasticleukemia (Ph+ ALL) who are resistant or intolerant to prior therapy.

Pharmaceutical compositions including TKIs, as well as dosage and routesof administration, are known in the art or can be determined usingroutine experimentation.

For example, the recommended starting dosage of dasatinib for adultswith chronic phase CML is 100 mg administered orally once daily (QD).The recommended starting dosage for accelerated phase CIVIL, myeloid orlymphoid blast phase CIVIL, or Ph+ ALL is 140 mg/day administered orallyonce daily. In clinical studies of adult CML and Ph+ ALL patients, doseescalation to 140 mg once daily (chronic phase CIVIL) or 180 mg oncedaily (advanced phase CML and Ph+ ALL) was allowed in patients who didnot achieve a hematologic or cytogenetic response at the recommendedstarting dosage.

Examples

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples describedherein.

Collection of Squamous Cell Lung Cancer Samples:

For the primary and secondary screens tumor samples were obtained undera general tissue collection protocol for patients with lung cancer whoare consented to tissue collection for research, including DNAsequencing, prior to surgery. All patients with resectable biopsy-provensquamous cell lung cancers (as diagnosed by a board-certified AnatomicPathologist) were eligible and eligible subjects underwent a detailedinformed consent procedure prior to enrollment on the protocol whichincluded a discussion of the use of tissue samples for DNA sequencingstudies and written documentation of consent. In addition, DNA samplesfrom de-identified squamous cell lung cancer patients were obtained fromthe Ontario Cancer Institute for the primary and secondary screens aspart of a Dana Farber/Harvard Cancer Center IRB-approved collection ofde-identified tumor samples for DNA sequencing studies. IRB approval forcollection of de-identified samples was subject to a review of the localIRB-approved protocols for all external sites to ensure an adequateinformed consent process had taken place.

The validation screen was performed as follows. Samples were collectedin accordance with a tissue collection protocol approved by theUniversity of Cologne Ethics Committee which involved a detailedinformed consent process including a discussion of genetic testing priorto the subject's surgery with written documentation of consent. Again,all patients with biopsy-proven squamous cell lung cancer were eligibleregardless of disease stage as long as their tumors were consideredresectable. De-identified samples were also collected from additionalEuropean sites including Haukeland University Hospital, UniversityHospital Zurich, Université Joseph Fourier, Oslo University Hospital,Jena University Hospital and University Medical Centre Groningen. At allsites samples were obtained in accordance with an IRB approved tissuecollection protocol and the collection of de-identified samples fromthese sites was approved by the University of Cologne Ethics Committeeafter a review of local collection protocols.

For the single patient sample obtained from the recent clinical trial ofcombination therapy with dasatinib and erlotinib for advanced lungcancer DNA was obtained under an IRB-approved protocol and thesequencing of the de-identified sample by both 454 and Sanger sequencingwas performed with approval after a review of the collection protocol.

In all instances specimens were continuously selected at the site ofsurgery in order to avoid sampling bias and all samples werede-identified prior to processing for DDR2 sequencing. When available,de-identified correlative clinical data was provided with the samples,though this data was not available to the investigators prior to samplegenotyping. Patients with a prior history of tumors involving a visceralorgan site were excluded to avoid the inclusion of metastases.

DDR2 Sequencing.

DDR2 was sequenced from genomic DNA obtained from squamous lung cancercell lines and patient samples by conventional Sanger sequencing. In thediscovery set 20 patient samples and matched normal DNA were used forsequencing 201 genes including 90 kinases. All mutations were verifiedas somatic. Mutations were identified using an automated mutation callerand then verified manually with comparison made to the matched normalsequence in the case of all primary tumor samples. In the secondaryscreen 35 additional patient samples and 13 SCC cell lines were used forsequencing the six mutated tyrosine kinases identified in the primaryscreen (DDR2, FGFR2, NTRK2, JAK2, CDK8 and FLT3). In the validationscreen 222 total samples underwent sequencing of the DDR2 gene. In allcases except D125Y matched normal DNA was available to verify themutation as somatic.

Cell Culture.

A549, NCI-H2286, HCC-366 and NCI-H1703 cells were obtained from the corecollection at the Dana Farber Cancer Institute, having previously beenpurchased from the ATCC and used to establish a collection of earlypassage lung cancer cell lines which were analyzed by fingerprinting andSNP arrays (32). All cells used for the experiments described in thismanuscript were obtained from freezes made at that time. Lung cancercell lines were grown in RPMI (Invitrogen) with 10% fetal calf serum,NIH-3T3 cells were grown in DMEM (Mediatech) with 10% serum and Ba/F3cells in RPMI supplemented with 10% serum and IL-3 (BD Biosciences) at10 ng/ml. For IL-3 withdrawal experiments Ba/F3 cells were collected viacentrifugation, washed once in sterile PBS and then resuspended in mediawithout IL-3. Colony formation assays in NIH-3T3 cells were performed insix-well plates in which 25,000 NIH-3T3 cells were plated in triplicatein 1 ml of 0.33% top agar on top of 2 ml of 0.5% bottom agar. Afterthree weeks colonies were counted using the NIH ImageJ software.

Vectors.

The full-length DDR2 cDNA was obtained from Origene and cloned into theEcoRI site of the retroviral vector p-Wzl-Blast and p-Babe-purofollowing the addition of a c-terminal FLAG tag by PCR. Mutants weregenerated by site-directed mutagenesis using the Quickchange sitedirected mutagenesis kit (Strategene). All mutations were verified bysequencing. sh-RNA lentiviral vectors for DDR2 were obtained from TheRNAi Consortium at the Broad Institute (46, 47). DDR2 sh-RNA-2corresponds to TRC clone TRCN0000121117 with hairpin sequence5′-CCGG-CCCATGCCTATGCCACTCCAT-CTCGAG-ATGGAGTGGCATAGGCATGGG-TTTTTG-3′(SEQ ID NO:2) targeting the 3′ UTR of DDR2. DDR2 sh-RNA-5 corresponds toTRC clone TRCN0000121121 with hairpin sequence5′-CCGG-CCCTGGAGGTTCCATCATTTA-CTCGAG-TAAATGATGGAACCTCCAGGG-TTTTTG-3′(SEQ ID NO:3) targeting the coding sequence of DDR2. Both hairpins wereprovided in the pLKO vector. A hairpin targeting GFP (shGFP) wasobtained from TRC as well and used as a control.

Viral Infections.

The DDR2 transgene was expressed in the lung cancer cell lines, NIH-3T3cells and Ba/F3 cells using retroviral transduction with the pWzlvector, as has been previously described. Briefly, 293T cells were usedto generate the virus with the appropriate pWzl or pBabe vector andpackaging vector transfected using Fugene (Roche). Cells were subjectedto two rounds of overnight infection in the presence of polybrene andstable cells generated using blasticidin selection at 10 mg/ml for 3T3,Ba/F3 and A549, 2 mg/ml NCI-H2286 and for NCI-H1703 and 1 mg/ml forHCC-366. Lentiviral infections were performed per the on-line TRCprotocol (48) with 293T cells transfected with the suggested threevector combination of pLKO, VSVG and delta 8.9. Virus was collected andused to infect the lung cancer cell lines for six hours in the presenceof polybrene. Stable cell lines were generated using puromycin selectionat a concentration of 2 mg/ml for NCI-H2286 and 4 mg/ml for NCI-H1703,A549 and HCC-366.

Cell Proliferation and Viability Assays.

Cell proliferation was measured with the Cell-Titer-Glo reagent(Promega) per the manufacturer's instructions. For experiments with theSCC cell lines cells were plated in clear-bottomed 96 well plates at adensity of 1500 cells per well. The following day the drug was added andcell proliferation was measured six days later for the SCC cell lines.For Ba/F3 cells were plated at 5000 cells per well and the drug addedthe same day. Proliferation was measured four days later. Proliferationmeasurements were made using a standard 96 well plate luminometer/platereader. Data are shown as relative values in which the luminescence at agiven drug concentration is compared to untreated cells of the same celltype. Kinase inhibitors were purchased from LC Labs or were synthesizedby Nathanael Gray's laboratory at Harvard Medical School. In vitro IC50sfor DDR2 were determined for all compounds by LanthaScreen TR-FRETkinase activity assays performed by Invitrogen. Cell viability wasmeasured using a vi-Cell reader to stain cells with trypan blue and togenerate 50 independent images for each measured sample. Annexin V (BDBiosciences) analysis was performed on dasatinib treated cells 48 hoursafter addition of drug per the manufacturer's protocol. For sh-RNAexperiments cells were plated at a density of 1500 cells per well in 96well plates following puromycin selection. Proliferation was measuredfour days later as compared to cells expressing a hairpin targeting GFP.

Immunoblots:

Immunoblots were performed using the Nupage system (Invitrogen) per themanufacturer's protocol. Cells were lysed in 1% NP-40 with protease(Roche) and phosphatase inhibitors (Calbiochem) and proteinconcentration assayed with the Bradford reagent (Bio-Rad). Primaryantibodies used were Flag-M2 (Sigma), phospho-Y417-Src (Cell SignalingTechnologies), phospho-Y694-STAT5 (Cell Signaling) and Actin (SantaCruz). A DDR2 antibody was generated for this project by Bethyl Labs.Secondary HRP-conjugated antibodies were all obtained from Pierce andproteins detected by pico-ECL (Thermo Scientific). Images were importedinto Adobe Illustrator using an Epson 4490 scanner. In some cases,brightness and/or contrast of the scanned images was adjusted forclarity and blots were cropped to display the area of interest in thedisplayed figures. In all cases adjustment of brightness or contrast theadjustment was applied uniformly to the image as a whole.

Xenografts:

All animal experiments were performed according to institutionalguidelines regarding animal safety. Nude mice were injected with thelung cancer cell lines at a density of 2.5 (A549), 3.0 (NCI-H1703) or5.0 (NCI-H2286 and HCC-366) million cells per injection to try tocontrol for the variable rates of tumor growth in the animals. Cohortsof ten mice were injected at three sites for each cell type and the micewere observed until the tumor volume approached 150 cubic millilitersfor A549 and NCI-H1703 or 100 cubic milliliters for NCI-H1703. At thattime mice were treated with dasatinib at 50 mg/kg or vehicle controldaily for two weeks and tumor size measured during the treatment period.

Statistics:

For proliferation and colony formation assays mean values from a minimumof triplicate samples are reported as well as standard errors ascalculated by Microsoft Excel. IC₅₀ values were obtained using Graph PadPrism software. Power and sample size calculations were performed usingthe Interactive Statistics Web Resource (43).

Example 1. DDR2 is Mutated in Squamous Cell Lung Cancer

Sanger sequencing of 201 genes, including the entire tyrosine kinome,was performed in an initial set of 20 primary lung SCC samples andmatched normal controls. Somatic missense mutations were identified in25 genes in this discovery sample set including six in tyrosine kinasegenes (FIG. 1a ). Recurrent somatic mutations were identified in TP53(n=8), and in the tyrosine kinase genes: Discoidin Domain Receptor 2(DDR2; n=2) and Kinase insert Domain Receptor (KDR; n=2) (FIG. 1a ).Subsequent sequencing of six of the mutated tyrosine kinase genes (DDR2,FGFR2, NTRK2, JAK2, FLT3 and CDK8), selected on the basis of beingpossible therapeutic targets, in a secondary screen of 48 squamous celllung cancer samples including 13 cell lines revealed four additionalDDR2 mutations (FIG. 1a ) as well as three FLT3 mutations, two NTRK2 andJAK2 mutations and one mutation in each of FGFR2 and CDK8.

Given that DDR2 was the most frequently mutated gene in the primary andsecondary screen, DDR2 was sequenced in a validation cohort of 222primary lung SCC samples which yielded an additional five samples withmutation, resulting in an overall frequency of 3.8% (n=11) in 290 totalsamples and an overall frequency of 3.2% in primary lung SCC sampleswhen cell lines were excluded (n=9/277) (FIG. 1a ). Mutations were foundboth in the kinase domain and in other regions of the protein sequenceand two mutations were identified at G774 (FIG. 1b ). The L239R andI638F mutations were identified in the HCC-366 and NCI-H2286 SCC celllines, respectively, and the remainder of the mutations was found inprimary SCC samples. The majority of the mutations resided in regions ofhigh degrees of amino acid conservation as compared to the murine,zebrafish and C. elegans homologs of DDR2 (FIG. 1c ). Additional genomicanalysis of previously reported copy number and gene expression datasetsdid not reveal any evidence of DDR2 overexpression in SCCs as comparedto normal lung or lung adenocarcinoma nor were any copy numberalterations in DDR2 found (19, 22-24). A query of the limited clinicalinformation accompanying the sequenced samples did not Identify anysignificant correlation of DDR2 mutation status with the age, sex orsmoking status of the patients.

Example 2. DDR2 Mutant Cell Lines are Selectively Sensitive to TyrosineKinase Inhibitors and to Sh-RNA-Mediated Depletion of DDR2

To assess whether targeting DDR2 might be a promising therapeuticstrategy in lung SCC, several tyrosine kinase inhibitors reported toinhibit DDR2 including imatinib and dasatinib, drugs which areFDA-approved for clinical use for targeting BCR-Abl in chronicmyelogenous leukemia and acute lymphoblastic leukemia, c-KIT ingastrointestinal stromal tumors and PDGFR in chronic myelomonocyticleukemia (21, 25-28) were analyzed. Fluorescence resonance energytransfer (FRET) measurements provided in vitro K_(d) values of dasatinib(5.4 nM) and imatinib (71.6 nM) for recombinant DDR2 (Table 1).

TABLE 1 Chemical structures and Kd for DDR2 for the compounds describedin the manuscript. Compound Structure Kd(nM) Dasatinib

5.4 Imatinib

71.6 Nilotinib

35.4 AZD0530

291 AP24534

8.99

Dasatinib showed particular efficacy against SCC cell lines bearing DDR2mutations, as dasatinib inhibited proliferation of the DDR2-mutantNCI-H2286 and HCC-366 cells with calculated IC_(50s) of 139 and 140 nMrespectively (FIG. 2a ). Of note, a recent pharmacokinetic analysis ofdasatinib in lung cancer patients demonstrated that peak concentrationsof dasatinib were in the range of 300 ng/ml (615 nM) at the maximumtolerated dose of 140 mg daily, a dose approved for use in leukemias(29). Imatinib was less potent when tested in the same cell lines withrespective IC_(50s) of 1.2 and 1.0 mM for the DDR2-mutant NCI-H2286 andHCC-366 cell lines (FIG. 2e ). Dasatinib and imatinib were lesseffective against the A549 cell line which is known to harbor a KRASmutation and does not have any DDR2 mutations (calculated IC₅₀ of 7.4 mMfor dasatinib and 2.3 mM for imatinib). Consistent with previousreports, the NCI-H1703 SCC cell line, which contains a PDGFRAamplification, was sensitive to both drugs, serving as a positivecontrol for our assay (30, 31). Notably, no other somatic mutations havebeen reported in the COSMIC database for NCI-H2286 or HCC-366 lines tosuggest alternative dasatinib targets and a previous report examiningthe drug sensitivities of 83 NSCLC cell lines identified HCC-366 as themost sensitive squamous cell lung cancer line to dasatinib, thoughNCI-H2286 and NCI-H1703 were not assayed (32). Treatment of the DDR2mutant cell lines with dasatinib appeared to lead to cell death asopposed to cell cycle arrest as measured by trypan blue exclusion (FIG.2f ). Dasatinib treatment was associated with an increase in cellularannexin V staining, suggesting that the treated cells died by apoptosis.

To validate DDR2 as a relevant target of dasatinib in SCCs a DDR2transgene with a threonine to methionine mutation at amino acid 654, amutation site shown previously to render DDR2 dasatinib-insensitive in amanner similar to the ability of the T790M mutation in EGFR to conferacquired resistance to the tyrosine kinase inhibitors erlotinib andgefitinib (33), was ectopically expressed. The dasatinib-insensitiveDDR2 “gatekeeper” mutant was introduced in cis with the observed L239Rand I638F mutations in the HCC-366 and NCI-H2286 cell lines respectivelyas well as alone in NCI-H1703. Expression of the gatekeeper mutation ledto a decrease in dasatinib sensitivity in both DDR2 mutant cell linesand had a modest effect on NCI-H1703 (FIG. 2b ; transgene expression isshown in FIG. 2i ). While the calculated IC₅₀ for NCI-H1703 did notchange with ectopic expression of the gatekeeper, the IC₅₀ increased by35-fold for NCI-H2286 and 209-fold for HCC-366, respectively.Interestingly, a parallel sequencing project in our lab identified aT654I mutation in DDR2 in a primary endometrial carcinoma sample.

Dasatinib was originally designed as an inhibitor of Src and is amulti-targeted tyrosine kinase inhibitor (34). Dasatinib treatment isassociated with toxicity in patients including myelosuppression and thedevelopment of pleural and pericardial effusions (35, 36). In an attemptto identify additional agents which could potently inhibit DDR2 withless associated toxicity we screened a panel of 20 tyrosine kinaseinhibitors which were predicted to have the potential to inhibit DDR2based on their respective structures. We found that nilotinib, asecond-generation BCR-Abl inhibitor, as well as with AP24534, a thirdgeneration BCR-Abl inhibitor which displays activity against BCR-Abl andimatinib-resistant BCR-Abl (36), inhibited the proliferation of SCClines harboring DDR2 mutations (FIGS. 2g and 2h ). We observed thatAP24534 treatment resulted in a greater degree of inhibition thannilotinib which was agreement with calculated in vitro K_(d) values of35.4 nM for nilotinib and 9.0 nM for AP24534 as compared to 5.4 nM fordasatinib (Table 1).

Example 3. Sh-RNAs Targeting DDR2 Kill DDR2-Mutant SCC Cell Lines

As an independent measure of DDR2-dependency short-hairpin RNAstargeting DDR2 were expressed using lentiviral vectors in the NCI-H2286,HCC-366 and NCI-H1703 cell lines. A set of sh-RNA-expressing plasmidswas screened for the ability to knock-down DDR2 mRNA expression byreal-time PCR in NCI-H2286 cells and selected two hairpins for furtheranalysis given their ability to reduce DDR2 mRNA levels by approximately50%. Knock-down of DDR2 by these two sh-RNAs led to a reduction inproliferation of the two DDR2 mutant cell lines but not ofPDGFRA-amplified NCI-H1703 cells which had been sensitive to imatiniband dasatinib in our proliferation assays (FIG. 2c ). The reduction inproliferation appeared to correlate with the degree of knock-down as theobserved phenotype was greater with sh-RNA-2 than sh-RNA-5 (FIG. 2c )and appeared to be caused by cell death and not cell cycle arrest.

To assess the specificity of the observed knock-down phenotype a similarexperiment was performed in NCI-H2286 and HCC-366 cells ectopicallyexpressing their described mutated forms of DDR2 (I638F and L239Rrespectively); endogenous DDR2 was knocked-down with sh-RNA-2 whichtargets the 3′ UTR of DDR2 and so would not be expected to interferewith ectopic expression of DDR2. For both NCI-H2286 and HCC-366, ectopicexpression of DDR2 attenuated the anti-proliferative effect ofendogenous DDR2 knock-down and the effect was of greater magnitude inNCI-H2286, perhaps due to a greater degree of off-target effects inHCC-366 (FIG. 2d ).

Example 4. DDR2 Mutations are Associated with Dasatinib Sensitivity InVivo

To analyze the effects of dasatinib treatment in a somewhat morephysiological setting, xenograft studies were performed in athymic nudemice in which cohorts of mice were injected with NCI-H2286, HCC-366,NCI-H1703 and A549 cells. HCC-366 cells did not form tumors in the miceand could not be analyzed further. Following tumor formation of thethree tested lines mice were treated with dasatinib at 50 mg/kg by oralgavage for two weeks or vehicle control. Dasatinib treatment led to adecrease in tumor size in the NCI-H1703 and NCI-H2286 lines but not inA549, consistent with the in vitro results (FIG. 3).

Example 5. DDR2 Mutations are Oncogenic and DDR2-Driven Transformationis Dasatinib-Sensitive

Next, the ability of DDR2 mutations to confer an oncogenicgain-of-function phenotype was examined. Ectopic expression of a subsetof the DDR2 mutants identified in our primary and secondary screens(n=2/6) promoted the formation of colonies in soft agar of NIH-3T3 cells(FIG. 4e ). Colony formation was greatest in the L63V and I638F mutantsat a level comparable to that driven by expression of thegain-of-function L858R mutation in EGFR and modest in the remainder ofthe genotypes. Colony formation could be inhibited with a singleapplication of dasatinib at the time of plating in the case of the L63Vmutant, the mutant which reproducibly formed the most colonies in ourassay (FIG. 4a ). Dasatinib treatment also inhibited the colonyformation of NIH-3T3 cells expressing the L858R mutation in EGFR,consistent with previous reports, and did so to a lesser extent inNIH-3T3 cells stably expressing the activating G12V KRAS mutation (FIG.4a ) (32, 37).

As the observed gain-of-function phenotype was modest for many of theDDR2 mutants in NIH-3T3 cells, the transforming potential of DDR2 wasevaluated in the interleukin-3 (IL-3)-dependent hematopoietic cell lineBa/F3. Ectopic expression of all six DDR2 mutants identified in theprimary and secondary screens led to IL-3-independent growth of Ba/F3cells as did high levels of expression of wild-type DDR2 and nodifferences were observed in the time to transformation or the rate ofIL-3 independent proliferation (FIG. 4f ). A kinase-dead DDR2 transgene(K608E) did not support the IL-3-independent growth of Ba/F3 cells (FIG.4f ). While culture with the less potent DDR2 inhibitor imatinib did notlead to significant killing of Ba/F3 cells expressing DDR2 mutations ascompared to cells grown in the presence of IL-3, culture with dasatinibled to cell death in all cell lines expressing DDR2 mutants with a meancalculated IC₅₀ of 680 nM for the mutants and 30 mM for the control(FIGS. 4b and 4g ). The third-generation BCR-Abl inhibitor AP24534 wasalso effective in killing the IL-3-independent Ba/F3 cells expressingmutant forms of DDR2, suggesting that this class of drugs may beeffective against DDR2-driven neoplasms while the second generationBCR-Abl inhibitor nilotinib demonstrated modest activity against theDDR2-tranformed Ba/F3 cells (FIGS. 4h and 4i ). Survival of Ba/F3 cellsin the absence of IL-3 was associated with maintenance of STAT5phosphorylation as has been previously shown (FIG. 4j )(38).

Example 6. DDR2 Transformed Cell Lines Maintain Src Phosphorylation andare Especially Sensitive to Dual Inhibition of DDR2 and Src

Given that the type I kinase inhibitor dasatinib was more potent inDDR2-transformed Ba/F3 cells than the more target-specific type IIinhibitors nilotinib and imatinib, whether the potency of dasatinib inthis system might be due to effects of dasatinib on other kinases inaddition to DDR2 was evaluated. DDR2 has previously been shown torequire Src for maximal kinase activity (16) and levels ofphosphorylated Src were maintained in Ba/F3 cells expressing DDR2mutants in the absence of IL-3 (FIG. 4j ). To test whether the abilityof DDR2 mutations to confer IL-3-independent proliferation in Ba/F3cells might depend on both DDR2 and Src activity, Ba/F3 cells expressingDDR2 were treated with AZD0530, a highly selective Src-family kinaseinhibitor which displays minimal activity against DDR2 as compared tothe other inhibitors described in this manuscript (in vitro K_(d) 291nM, Table 1) (39). Similar to nilotinib treatment, AZD0530 had a modesteffect on the proliferation of the IL-3-independent DDR2-expressingBa/F3 cells (FIGS. 4i and 4k ). However, when Ba/F3 cells expressing theL63V DDR2 mutation were grown in 50 nM nilotinib, a concentrationassociated with little effect on proliferation of wild-type Ba/F3 cellsor Ba/F3 cells expressing DDR2 mutations (FIG. 4i ), the addition ofAZD0530 led to a marked reduction in proliferation of Ba/F3 cellsexpressing DDR2 L63V, suggesting that the coordinated activity of DDR2and Src-family kinases may be required for the ability of DDR2 mutatedBa/F3 cells to grow in the absence of IL-3 and thereby providing apossible explanation for the potency of dasatinib in this system (FIGS.4c and 4m ). A similar additive effect of AZD0530 was observed when theBa/F3 cells were co-treated with AZD0530 and 50 nM of either AP24534 ordasatinib (FIG. 4c for L63V; for a more detailed version of thisexperiment including additional DDR2 mutants see FIGS. 4m-o ). AZD0530reduced Src and STAT5 phosphorylation in a dose-dependent fashion in theDDR2 L63V-expressing Ba/F3 cells when used as a single agent or incombination with nilotinib, AP24534 or dasatinib (FIGS. 4d and 4l ).

Example 7. Observation of a DDR2 Kinase Domain Mutation in a ClinicalTrial Subject with a Radiographic Response to Combination Therapy withDasatinib and Erlotinib

Two recent early-phase clinical trials of dasatinib have been reportedin which subjects with advanced stage lung cancer were treated witheither dasatinib or a combination of dasatinib and erlotinib (29, 40).One of seven subjects with a squamous cell lung cancer exhibited asignificant shrinkage in tumor size while undergoing therapy with acombination of dasatinib and erlotinib, and unlike the other subject onstudy with lung adenocarcinoma who exhibited a response to treatment,there was no evidence of EGFR mutation in the subject with squamous celllung cancer. The patient was a 59 year old Caucasian woman with a ⅓ packper day smoking history for 38 years who quit one year before herdiagnosis of lung cancer. She was found to have a left lower lobe stageI (T2N0M0) squamous cell lung cancer and received primary treatment withweekly carboplatin and paclitaxel with concomitant 70 Gy of radiationresulting in a complete response. However, approximately one year latershe developed progression of disease within the radiation field andtreatment was initiated with s standard dose carboplatin and paclitaxelwithout response. She then began combination dasatinib and erlotinibtherapy on protocol. A restaging CT scan after nearly 2 months indicatedtumor shrinkage and the patient experienced improved symptoms (resolveddyspnea and cough) (FIGS. 5A and 5B). She remained on treatment for 14months until therapy had to be discontinued secondary totreatment-induced airspace disease and pleural effusions.

Directed sequencing of DDR2 was performed in a pre-treatment tumorspecimen derived from this individual and a novel DDR2 kinase domainmutation, S768R, was identified that was present in 844 of 3020 (28%) ofreads obtained by 454 sequencing and independently verified by Sangersequencing. The mutation could not be verified as somatic as no normalDNA was available for this individual who is deceased. There were noother SCC subjects who responded to therapy on this study or asubsequent study of dasatinib alone (n=13 total) to further explore thiscorrelation.

Three-dimensional modeling of the S768R mutation in the context of theDDR2 kinase domain was performed. A structural model of the kinasedomain of DDR2 (residues 545-854) was generated based on the crystalstructure of the Abl kinase domain in the active (DFG-in) conformation(PDB code: 3DK6) in complex with a small molecule inhibitor. Dasatinibwas modeled into the ATP-binding site of DDR2 based on the crystalstructure of dasatinib in complex with cSrc (PDB code: 3G5D). Theproposed binding mode showed the inhibitor core within hydrogen bondingdistance to the backbone of the hinge region of the kinase domain. Theterminal ethanol-piperazine was solvent exposed. The activating mutationSer768Arg was found at the N-terminal end of a helix below the 5activation loop. The structural model indicated that the side chain ofSer768 in wild type DDR2 likely resides in a packed environment flankedby Glu213 and Phe220. The extra charge and steric bulk of an Arg atposition 768 in mutant DDR2 is likely to introduce structural changes inthis region which could potentially alter the kinase activity and/orregulatory mechanisms of DDR2. These results suggest that the S768Rsubstitution is likely to alter the kinase activity of DDR2.

REFERENCES

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of treating a human subject diagnosed with squamous cellcarcinoma (SCC) of the lung, wherein the subject has a sequence in adiscoidin domain receptor 2 (DDR2) gene encoding at least one amino acidvariation relative to a reference amino acid sequence, and wherein theat least one amino acid variation is L63V, I120M, D125Y, G253C, G505S,C580Y, T765P, G774V, or G774E, the method comprising administering atreatment comprising a tyrosine kinase inhibitor (TKI).
 2. (canceled) 3.The method of claim 1, wherein the subject is human and the referenceamino acid sequence comprises SEQ ID NO:
 1. 4. The method of claim 1,wherein the reference sequence is obtained from non-cancerous cells ofthe same subject. 5.-7. (canceled)
 8. The method of claim 1, comprisingdetermining a nucleic acid sequence of a coding region of a discoidindomain receptor 2 (DDR2) gene.
 9. The method of claim 1, wherein the atleast one amino acid variation results in a decrease in expressionlevels, half-life, or kinase activity of the DDR2 protein.
 10. Themethod of claim 1, wherein the TKI is dasatinib, nilotinib, imatinib, orponatinib.
 11. The method of claim 10, wherein the TKI is dasatinib.12.-20. (canceled)
 21. The method of claim 1, further comprisingidentifying the subject as having at least one of said amino acidvariations relative to the reference amino acid sequence.
 22. The methodof claim 21, wherein identifying the subject comprises performing anassay to determine a nucleic acid sequence of all or part of a DDR2 genein a sample comprising nucleated cells from a SCC in the subject,wherein the assay comprises contacting the DDR2 gene with a nucleic acidprobe that specifically hybridizes with a sequence encoding at least oneamino acid variation relative to a reference amino acid sequence, andwherein the at least one amino acid variation is L63V, I120M, D125Y,G253C, G505S, C580Y, T765P, G774V, or G774E; and detecting in the samplethe nucleic acid sequence encoding at least one amino acid variation ofL63V, I120M, D125Y, G253C, G505S, C580Y, T765P, G774E, or G774V in theDDR2 gene.